siRNA (small interfering RNA) is a double-stranded RNA with 19 to 21 base pairs, and is a molecule playing a major role in RNA interference (RNAi), which is known as potent and specific gene expression suppression phenomenon (Fire A et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature, vol. 391, pp. 744-745, 1998). Since RNAi in mammalian cells was reported by Tushl et al. in 2001 (Sayda M et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, vol. 411, pp. 494-498, 2001), research and development for the purpose of application of siRNA to every treatment of disease have been promoted. However, in the preceding development, sites in which affected areas can be easily reached, such as eyes and respiratory organs, have been targeted, and therefore the range of application is limited. siRNA is an unstable chemical species and is easily degraded under physiological circumstances. It is known that, when siRNA is solely administered intravenously to a mouse, it is excreted from kidney rapidly (t1/2=several minutes). Therefore, DDS, which improves disposition of siRNA, is desired as a key to practical application of siRNA.
However, systemic DDS is still at the level of laboratory and miles away from practical application. Even in the case of the system of SNALP (Stable Nucleic Acid Lipid Particles) provided by Alnylam and Protiva Biotherapeutics, regarding which the most advanced achievement has been reported, it is difficult to apply the system to tissue other than liver tissue due to passive uptake into liver (Tracy S et al., RNAi-mediated gene silencing in non-human primates, Nature, vol. 441, pp. 111-114, 2006). Meanwhile, in terms of long-term retention in the blood, retentivity was successfully and dramatically improved by the system of Wrapped liposome provided by Kyowa Hakko, though single-stranded DNA was targeted by the system (Masahiro Yamauchi et al., Improved formulations of Antisense oligonucleotides using wrapped liposomes, J. Control Release, vol. 114, pp. 268-275, 2006), and future development regarding siRNA is particularly expected. However, in the case of liposome, it is often difficult to release an agent therein since it is too stable.
As described above, in order to extend the range of application of siRNA, it is extremely important to provide a technique for improving disposition of siRNA and allowing siRNA administration into the blood stream.
With respect to plasmid DNA and antisense DNA, nanometer-scale structures in which such a DNA is encapsulated using poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys) (i.e., polymer micelles) have been formed, and their utility has been reported (K. Kataoka et al., Spontaneous formation of polyion complex micelles with narrow distribution form antisense oligonucleotide and cationic block copolymer in physiological saline, Macromolecules, vol. 29, pp. 8556-8557, 1996; M. A. Woflert et al., Characterization of vector for gene therapy formed by self-assembly of DNA with synthetic block co-polymers. Hum., Gene Ther., vol. 7, pp. 2123-2133, 1996; S. Katayose et al., Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer. Bioconjug., Chem., vol. 8, pp. 702-707, 1997; S. Katayose et al., Remarkable increase in nuclease resistance of plasmid DNA through supramolecular assembly with poly(ethylene glycol)-poly(L-lysine) block copolymer, J. Pharm. Sci., vol. 87, pp. 160-163, 1998).
Such a polymer micelle is formed by self-assembly caused by electrostatic interaction between a polycation portion in the above-described block copolymer (a polylysine portion in PEG-PLys) and a polyanion, a nucleic acid molecule (DNA, etc.). The polymer micelle formed has a core-shell-type structure, in which the polyion complex portion between polycation and polyanion is an inner core-like portion, and the surface layer thereof is like being covered with polyethylene glycol (PEG). Therefore, it is expected that the polymer micelle can avoid the foreign substance recognition mechanism in the body and renal excretion.
In consideration of the fact that the above-described plasmid DNA and antisense DNA are analogs of siRNA, the present inventors tried to form a siRNA-encapsulated polymer micelle using PEG-PLys as a strategy to improve disposition of siRNA. However, the product actually obtained had low structural stability and did not have a sufficient core-shell-type micelle structure of interest, and its ability to deliver siRNA into a culture cell was extremely low. Therefore, it was extremely difficult to utilize PEG-PLys as a carrier of siRNA.